This proposal describes a multifaceted plan to advance our understanding of the regulation of alternative RNA splicing in the nervous system. Alternative splicing is a major driving force generating proteomic diversity in mammals, yet its mechanisms, regulatory networks, and responsiveness to neuronal function and morphogenesis are poorly understood. The nervous system is a rich source of tissue-specific alternative splicing events, which alter the functions of proteins involved in cell communication and connection patterns. Biochemical approaches will be used in Aim 1 to characterize the mechanisms involved in the brain region-specific splicing of the CI and NI cassette exons of the NMDA R1 receptor transcript. Related experiments will determine the underlying basis for the positive and negative roles of the RNA binding protein, NAPOR, in these mechanisms, and will determine the roles of collaborating factors, such as PTB and hnRNP K. RNAi gene silencing approaches and primary neuronal cultures will be used in Aim 2 to characterize the roles of NAPOR and PTB in the splicing of transcripts involved in synaptic functions. Complementary experiments will examine the effects of the RNAi treatments on NMDA receptor function in neurons. Additional experiments in this aim will monitor the behavior of alternative splicing patterns in the primary cortical cultures in response to changes in ion channel activity, and during the course of neuronal differentiation. NAPOR-deficient transgenic mice will be generated in Aim 3 to determine the functional roles of its protein products at the level of splicing. Effects on development, growth and behavior will also be characterized. NAPOR-deficient mice will be applied toward a large-scale analysis of alternative splicing in the brain and in primary cultures. These experiments will involve large-scale mRNA and protein expression profiling to identify the spectrum of transcripts involved in NAPOR-dependent splicing pathways. Abnormalities in alternative splicing are associated with neurodegenerative, psychiatric and neuromuscular diseases, as well as various cancers. This research project will advance our understanding of the underlying mechanisms and plasticity of neuron-specific splicing, and provide insights into how these mechanisms fail in human disease.